• Journal of Microbiology and Biotechnology
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• v.33 no.11
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• pp.1457-1466
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• 2023

Enteric fever is caused by typhoidal Salmonella serovars (Typhi, Paratyphi A, Paratyphi B, and Paratyphi C). Owing to the importance of Salmonella serovars in clinics and public hygiene, reliable diagnostics for typhoidal serovars are crucial. This study aimed to develop a novel diagnostic tool for typhoidal Salmonella serovars and evaluate the use of human blood for clinically diagnosing enteric fever. Five genes were selected to produce specific PCR results against typhoidal Salmonella serovars based on the genes of Salmonella Typhi. Heptaplex PCR, including genetic markers of generic Salmonella, Salmonella enterica subsp. enterica, and typhoidal Salmonella serovars, was developed. Typhoidal Salmonella heptaplex PCR using genomic DNAs from 200 Salmonella strains (112 serovars) provided specifically amplified PCR products for each typhoidal Salmonella serovar. These results suggest that heptaplex PCR can sufficiently discriminate between typhoidal and non-typhoidal Salmonella serovars. Heptaplex PCR was applied to Salmonella-spiked blood cultures directly and provided diagnostic results after 12- or 13.5-h blood culture. Additionally, it demonstrated diagnostic performance with colonies recovered from a 6-h blood culture. This study provides a reliable DNA-based tool for diagnosing typhoidal Salmonella serovars that may be useful in clinical microbiology and epidemiology.

• The Plant Pathology Journal
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• v.29 no.4
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• pp.357-363
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• 2013

The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. nashicola were identified by PCR, after which two pairs of sequence characterized amplified region (SCAR) primers were designed from the nucleotide sequences of the markers. The SCAR primer pairs, designated as D12F/D12R and E11F/E11R, amplified 535-bp and 525-bp DNA fragments, respectively, only from genomic DNA of V. nashicola. The specificity of the primer sets was tested on strains representing three species of Venturia and 20 fungal plant pathogens. The nested PCR primer pair specific to V. nashicola was developed based on the sequence of the species-specific 525-bp DNA fragment amplified by primer set E11F/E11R. The internal primer pair Na11F/Na11R amplified a 235-bp fragment from V. nashicola, but not from any other fungal species tested. The nested PCR assay was sensitive enough to detect the specific fragment in 50 fg of V. nashicola DNA.

• Korean Journal of Medicinal Crop Science
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• v.18 no.3
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• pp.173-179
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• 2010

This study describes an efficient approach to the development of DNA markers for use in distinguishing the Scrophularia species that have been used as useful medicinal crops. In order to distinguish Scrophularia species, DNA sequences of rpl-5 region in mitochondrial DNA of Scrophularia species were analysed for detecting sequence variations, and the PCR-RFLP method was applied for developing practicable DNA marker patterns. Several DNA variations were detected by the sequence comparison of rpl-5 region among Scrophularia species. Genetic relationship analysis of Scrophularia species was carried out based on these DNA variations. DNA variations of rpl-5 region were revealed that it was significantly efficient in genetic relationship analysis of Scrophularia species. In addition, Scrophularia species tested in this study were completely discriminated by four polymorphic genotypes by PCR-RFLP combined with Tsp509 I (^AATT) restriction enzyme. Our results suggested that DNA sequence variations of rpl-5 region were sufficiently useful for genetic relationship analysis of Scrophularia species. Polymorphic genotypes by PCR-RFLP using the Tsp509 I enzyme will be useful for discrimination of Scrophularia species as a practicable DNA markers.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.46 no.4
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• pp.341-343
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• 2001

Molecular markers have become fundamental tools for crop genome study. The objective of this study was to construct a genetic linkage map for cowpea with PCR-based molecular markers. Five hundred and twenty random RAPD primers were screened for parental polymorphism. Ninety RAPD markers from sixty primers was segregated in 75 F2 mapping population derived from the cross of local cultivars GSC01 and GSC02. 70 RAPD markers were found to be genetically linked and formed 11 linkage groups. Linkage map spanned 474.1 cM across all 11 linkage groups. There are six linkage groups of 40 cM or more, and five smaller linkage groups range from 4.9 to 24.8 cM. The average linkage distance between pairs of markers among all linkage groups was 6.87 cM. The number of markers per linkage group ranged from 2 to 32. The longest group 1 spans 190.6 cM, while the length of shortest group 11 is 4.9 cM. This map is further needed to be saturated with the various markers such as RFLP, AFLP, SSR and more various populations and primers. In addition, morphological markers and biochemical markers should be united to construct a comprehensive linkage map.

• Journal of Life Science
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• v.31 no.1
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• pp.66-72
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• 2021

This study developed a species identification method for the salted opossum shrimp of Acetes japonicus, A. chinensis (Korea, China), A. indicus (I, II), and Palaemon gravieri based on PCR-RFLP markers. Genomic DNA was extracted from the salted opossum shrimp. The COI gene was used to amplify 519 base pairs (bp) using specific primers. The amplified products were digested by Acc I and Hinf I, and the DNA fragments were separated by automated electrophoresis for RFLP analysis. When the amplified DNA product (519 bp) was digested with Acc I, A. japonicus, A. chinensis (Korea), and A. indius (II) showed two fragments, whereas a single band of 519 bp was detected in A. chinensis (China) and A. indius (I). Also, in the RFLP patterns digested by Hinf I, A. chinensis (Korea) and A. chinensis (China) showed a single band of 519 bp, while two fragments were observed in A. japonicus and A. indius (I) and four fragments in A. indius (II). The PCR amplicon of P. gravieri was digested by Acc I into 3 bands of 271, 202, and 46 bp and by Hinf I into a single band of 519 bp. Therefore, salted opossum shrimp-specific RFLP markers showing distinct differences between four species and two sub-species by PCR-RFLP analysis. Thus, the PCR-RFLP markers developed in this study are a good method for identifying the six types of salted opossum shrimp.

• Preventive Nutrition and Food Science
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• v.3 no.2
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• pp.198-202
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• 1998

The arbitrary primed polymerase chain reaction (AP-PCR) has been used to detect the genetic alternations in the related species. Simple and reproducible fingerprints of complex genomes can be generated using single arbitrary chosen primers and the PCR. The technique was applied to the Oryza species and characterized the relationship among three cultivars of rice species based on theresult of genomic DNA fingerprints. The results indicated that the polymorphism revealed in rice strains and the differences in the PCR product pattern could be represented for each strainis. There was many variationsin the PCR product pattern between cv. Dongin(japonica type)and cv.Hyangdo (indica type), and our chosen AP-primers can ge as markers for strain identification and verfication.

• The Plant Pathology Journal
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• v.36 no.5
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• pp.418-427
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• 2020

Xanthomonas campestris pv. campestris (Xcc), the pathogen of black rot which is the most destructive disease of Brassica vegetables throughout the world. Here, we reported two novel sequence-characterized amplified region (SCAR) markers (i.e., XccR6-60 and XccR6-67) for the detection of Xcc race 6 via re-alignment of the complete genome sequences of Xcc races/strains/pathovars. The specificity of SCAR primer sets was verified by mean of PCR amplification using the genomic DNA template of Xcc races/strains/pathovars and two other plant infecting bacterial strains. The PCR result revealed that the XccR6-60 and XccR6-67 primer sets amplified 692-bp and 917-bp DNA fragments, respectively, specifically from race 6, while no visible amplification was detected in other samples. In addition, the SCAR primers were highly sensitive and can detect from a very low concentration of genomic DNA of Xcc race 6. However, the complete genome sequence of Xcc race 6 is not yet publicly available. Therefore, the cloning and sequencing of XccR6-60 and XccR6-67 fragments from race 6 provide more evidence of the specificity of these markers. These results indicated that the newly developed SCAR markers can successfully, effectively and rapidly detect Xcc race 6 from other Xcc races/strains/pathovars as well as other plant pathogenic bacteria. This is the first report for race-specific molecular markers for Xcc race 6.

• Korean Journal of Medicinal Crop Science
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• v.19 no.3
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• pp.162-169
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• 2011

The rhizomes and herbal medicines originating from Acorus gramineus, A. calamus, A. tatarinowii, and A. gramineus var. pusilus, show significant similarity, and the correct identification of species is very difficult. Random Amplified Polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were used to develop a reliable method for identification of these four species. Several distinct SCAR markers were developed from species-specific RAPD amplicons for each species. Furthermore, a useful molecular marker was established for multiplex-PCR, in order to the four species could be distinguished concurrently. These markers allow efficient and rapid identification of closely-related Acorus species and will be useful for standardization of herbal medicines.

• The Korea Journal of Herbology
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• v.37 no.4
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• pp.9-16
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• 2022

Objectives : In the Korean Pharmacopoeia 12th edition (KP 12) and the Korean Herbal Pharmacopoeia (KHP), two authentic herbal medicines are described, namely Bang-gi (Cheong-pung-deung) and Mok-bang-gi, respectively. In China, Bun-bang-gi is also used as herbal medicine. This study was conducted to develop a molecular authentication tool for distinguishing the three herbal medicine used as Bang-gi, which are Sinomeni Caulis et Rhizoma (Rhizome of Sinomenium acutum), Stephaniae Tetrandrae Radix (Root of Stephania terandra), and Cocculi Radix (Root of Cocculus trilobus). Methods : Twelve samples of three species (four samples of S. acutum, five samples of S. tetrandra, and three samples of C. trilobus) were collected from different habitats. The sequences of internal transcribed spacer (ITS) regions were obtained and comparatively analyzed to design the species-specific sequence characterized amplified region (SCAR) primers. The specificity of each pair of SCAR primers that amplified species-specific amplicon was evaluated for establishing the singleplex and multiplex PCR assay tools. Results : The singleplex SCAR markers show discriminability in C. acutum, S. tetrandra, and C. trilobus. These SCAR markers were also efficiently authenticated three species in the multiplex SCAR amplification using single PCR reaction. Furthermore, these PCR assay methods were applicable to authenticate dried herbal medicines distributed in the markets. Conclusions : The SCAR markers and PCR assay tools help discriminate the three herbal medicines used as Bang-gi at the species levels and provide a reliable genetic method to prevent the inauthentic distribution of these herbal medicines.

• Weed & Turfgrass Science
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• v.2 no.2
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• pp.191-197
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• 2013

Two medium-leaf ecotypes (CY6069, CY6097) belonging to one species (Zoysia japonica) of Korean lawngrasses were selected in sod production fields in Jang Seong, Korea. They were reported to have distinct morphological and growth rate characteristics different from the preferred medium-leaf type zoysiagrass in Korea. This study was conducted to define further the genotypic difference at the molecular level and to develop DNA marker based on the specific DNA fragment. Polymorphic DNA fragments were first explored by using randomly amplified polymorphic DNA (RAPD) primers, which were then converted into PCR-based sequence characterized amplified region (SCAR) markers. The CY6069-specific primer set amplified about 550 bp successfully, while the CY6097 marker produced the expected 690 bp band, by which those markers were nominated by CY6069_550 and CY6069_690 SCARs, respectively. Together with the reported morphological and other phenotypic features, the SCAR markers confirmed in this study will be useful to identify those medium-leaf zoysiagrass genotypes when they are cultivated with other vegetatively propagated warm-season turfgrasses in sod farms.